Basic monocarboxyamide derivatives of actagardine having antibiotic activity and compositions thereof

ABSTRACT

The present invention is directed to a class of basic monocarboxyamide derivatives of actagardine. Actagardine (INN) is an antibiotic substance produced by actinoplanes strains such as Actinoplanes Sp. ATCC 31048 and Actinoplanes Sp. ATCC 31049 which are described in U.S. Pat. No. 4,022,884. Actagardine shows antimicrobial in vitro and in vivo activity against gram-positive organisms. Its complete chemical structure is not yet known but there is only information on its chemical functions and main fragments. In particular, it has been found that actagardine has two carboxylic functions and a primary amino function and can therefore be represented as follows:  &lt;IMAGE&gt;  The compounds of the invention are monoamide derivatives at one of the carboxy functions of actagardine. More particularly, they are basic monoamide derivatives of actagardine which may be schematically represented by the following formula I:    &lt;IMAGE&gt; Formula I  The compound of the invention possess improved antimicrobial activity in particular against gram positive bacteria.

The present invention is directed to a class of basic monocarboxyamidederivatives of actagardine. Actagardine (INN) is an antibiotic substanceproduced by actinoplanes strains such as Actinoplanes Sp. ATCC 31048 andActinoplanes Sp. ATCC 31049 which are described in U.S. Pat. No.4,022,884.

Actagardine shows antimicrobial in vitro and in vivo activity againstgram-positive organisms. Its complete chemical structure is not yetknown but there is only information on its chemical functions and mainfragments. In particular, it has been found that actagardine has twocarboxylic functions and a primary amino function and can therefore berepresented as follows: ##STR3##

The compounds of the invention are monoamide derivatives at one of thecarboxy functions of actagardine. More particularly, they are basicmonoamide derivatives of actagardine which may be schematicallyrepresented by the following formula I: ##STR4## wherein ##STR5##represents the actagardine nucleus, R represents the group ##STR6## R¹and R² independently represent hydrogen, a group of formula ##STR7## inwhich n represents an integer from 2 to 8 and R³ and R⁴ independentlyrepresent hydrogen or (C₁ -C₄) alkyl or R³ and R⁴ taken togetherrepresent a --(CH₂)₃ --, --(CH₂)₄ --, (CH₂)₂ --O--(CH₂)₂, --(CH₂)₂--S--(CH₂)₂ -- or --(CH₂)₅ -- group, or R¹ and R² taken together withthe adjacent nitrogen atom represent a piperazine mojety which may besubstituted in position 4 with a substituent selected from (C₁-C₄)alkyl, (C₅ -C₇)-cycloalkyl, pyridyl, benzyl, and substituted benzylwherein the phenyl mojety bears 1 or 2 substituents selected fromchloro, bromo, nitro, (C₁ -C₄)alkyl and (C₁ -C₄)alkoxy, R⁵ representshydrogen, (C₁ -C₄)alkyl or (C₁ -C₄)alkoxy (C₂ -C₄)alkyl, R.sup. 6represent hydrogen or (C₁ -C₄)alkyl with the proviso that R¹ and R² cannot simultaneously represent hydrogen, and the acid and base additionsalts thereof.

The term "(C₁ -C₄)alkyl" represents straight or branched alkyl chains offrom 1 to 4 carbon atoms, such as: methyl, ethyl, propyl, 1-methylethyl,butyl, 1-methylpropyl or 1,1-dimethylethyl while the term "C₂ -C₄)alkyl"represents straight or branched alkyl chains of from 2 to 4 carbon atomssuch as: ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl or1,1-dimethylethyl. The term "(C₅ -C₇)cycloalkyl" represents a cycloalkylgroup selected from cyclopentyl, cyclohexyl and cycloheptyl.

The term "(C₁ -C₄)alkoxy" represents a straight or branched alkoxy chainof 1 to 4 carbon atoms such as methoxy, ethoxy, propoxy, 1-methylethoxy,butoxy, 1-methylpropoxy and 1,1-dimethylethoxy. The compounds of theinvention possess acid and basic functions capable of forming salts.These salts can be prepared according to techniques well known in theart.

Examples of acid addition salts of the invention are: hydrohalides, suchas the hydrochloride and hydrobromide, sulfate, phosphate, nitrate,acetate, citrate, aspartate, methanesulfonate and toluenesulfonate.

Examples of salts with bases include the alkali metal and alkaline earthmetal salts such as the sodium, potassium, lithium, magnesium, zinc, andcalcium salts. The transformation of the non-salt form of a compound ofthe invention in a salt thereof, by adding the selected base or acid,and the reverse i.e. the transformation of an addition salt of acompound of the invention into the non-salt form, are within ordinarytechnical skill and are encompassed by the present invention.

In view of the similarity of the properties of the compounds of formulaI and their salts, what is said in the present application when dealingwith the biological activities of the compounds of formula I appliesalso to their pharmaceutically acceptable salts, and vice versa.

Representative examples of the derivatives of the invention are theactagardine derivatives wherein the monoamide mojety has the formula--COR wherein R represents:

    __________________________________________________________________________    NH(CH.sub.2).sub.2 NH.sub.2 ;                                                                     NH(CH.sub.2).sub.3 NH.sub.2                               NH(CH.sub.2).sub.4 NH.sub.2 ;                                                                     NH(CH.sub.2).sub.3 NHCH.sub.3                             NH(CH.sub.2).sub.3 N(CH.sub.3).sub.2 ;                                                            NH(CH.sub.2).sub.3 N(C.sub.2 H.sub.5).sub.2               NH(CH.sub.2).sub.3 N(C.sub.3 H.sub.7).sub.2 ;                                                     NH(CH.sub.2).sub.3 N(C.sub.4 H.sub.9).sub.2               NH(CH.sub.2).sub.5 N(CH.sub.3).sub.2 ;                                                            NH(CH.sub.2).sub.6 N(CH.sub.3).sub.2                      NH(CH.sub.2).sub.6 NHCH.sub.3 ;                                                                   N[(CH.sub.2).sub.2 NH.sub.2 ].sub.2                       N[(CH.sub.2).sub.3 NH.sub.2 ].sub.2 ;                                                             N[(CH.sub.2).sub.2 N(CH.sub.3).sub.2 ].sub.2              N[(CH.sub.2).sub.3 N(CH.sub.3).sub.2 ].sub.2 ;                                                    N[(CH.sub.2).sub.4 NH.sub.2 ].sub.2                        ##STR8##                                                                                          ##STR9##                                                  ##STR10##                                                                                         ##STR11##                                                 ##STR12##                                                                                         ##STR13##                                                 ##STR14##                                                                     ##STR15##                                                                                         ##STR16##                                                 ##STR17##                                                                                         ##STR18##                                                 ##STR19##                                                                                         ##STR20##                                                 ##STR21##                                                                                         ##STR22##                                                 ##STR23##                                                                                         ##STR24##                                                 ##STR25##                                                                                         ##STR26##                                                __________________________________________________________________________

The monoamide derivatives of the invention are prepared by reactingactagardine with a 2 to 6-fold molar excess of the selected amine offormula HNR¹ R², wherein R¹ and R² are as defined above, in a suitableinert organic solvent such as dimethylformamide (DMF) at a temperaturebetween 0° C. and room temperature and in the presence of a suitablecondensing agent.

When the amine R¹ R² NH contains a further primary amino group it shouldbe protected, as known in the art, in order to get the desired product.Obviously, a deprotection step is then necessary to obtain the desiredfinal product.

Representative examples of condensing agents are (C₁ -C₄)alkyl or phenylphosphorazidates such as, diphenyl phosphorazidate (DPPA), diethylphosphorazidate, di(4-nitrophenyl) phosphorazidate, dimorpholylphosphorazidate and diphenyl phosphorochloridate. The preferredcondensing agent is diphenyl phosphorazidate (DPPA).

A preferred molar excess of the selected amine over actagardine is from3 to 5 mole of amine per mole of actagardine, while the preferred molarexcess is a 4-fold molar excess.

The condensing agent is generally present in a slight molar excess,preferably from 1.1 to 3 mole per mole of actagardine.

The reaction temperature is preferably from 0° C. to 25°-30° C. Thepreferred temperature range is from 0° C. to 5° C.

The reaction can be monitored by TLC or HPLC. A preferred TLC techniqueincludes the use of silica gel plates (such as Silica-gel F₂₅₄ plates,Merck) with a mixture CH₃ CN:0.1M phosphate buffer pH 7.0, at a ratio of75 to 25 (v/v) for the piperazinyl derivatives, and at a ratio 60 to 40(v/v) for the others as the developing mixture. The spots may bedetected by both UV light at 254 nm and by charring with conc. H₂ SO₄ at120° C.

When the amine R¹ R² NH is reacted as a corresponding salt, e.g. thehydrochloride, it is necessary to add a suitable base in at least amolar proportion to obtain the free base of the amine R¹ R² NH whichreacts with actagardine. In this case, an excess of the base isgenerally preferred. Examples of bases suitable to free the amine R¹ R²NH from its salts include tertiary amine such as triethylamine ortrimethylamine, picoline and the like. As already said, when the amineR¹ R² NH contains a further primary amino group, it is necessary toprotect it before reacting it with actagardine. The protection of theprimary amino group (or groups) is made according to known per setechniques. A preferred way of protecting the primary amino function isby reaction with benzaldehyde in ethanol at room temperature to form thebenzylidene derivative which is then reacted with actagardine asdescribed above. Once the reaction is completed, the protecting groupcan easily be removed for example by treating with diluted hydrochloricacid at room temperature.

In so doing, a compound of formula I is obtained wherein R is asdefined, and R⁵ and R⁶ are hydrogen atoms or the corresponding salts.

The acid-base titration of these basic monoamide derivatives show thatindeed only one amidic bond is formed while the other carboxylicfunction of actagardine remains unreacted. This method of forming theamide derivative invention is therefore a selective method for preparinga mono-amide derivative of actagardine without forming the possiblediamide derivative.

The compound of the invention wherein R⁵ and/or R⁶ are as defined abovebut different from hydrogen are prepared by reacting the correspondingcompound wherein R⁵ and/or R⁶ are hydrogen with a suitable esterifyingor alkylating agent.

Suitable esterifying agents are acidic mixture of the selected alcoholof formula R⁵ OH. The reaction is generally conducted in an excess ofthe alcoholic solution which acts also as the reaction solvent. Thetemperature is in general about room temperature but temperaturesbetween 5° C. and 40° C. may be used. The reaction time varies dependingon the other reaction parameters, but in general the reaction iscompleted in 4-48 h. The reaction is in any case monitored by TLCprocedures which employ polar mixtures such as methanol/phosphate bufferpH 7, 7:3 (v/v) or butanol/acetic acid/water, 4:1:1 (v/v) and UVvisualization at 254 nm or carbonization at 120° C. with conc. H₂ SO₄.

The alkylation of the primary amino group of actagardine is preferablycarried out by reductive alkylation using the corresponding carbonyliccompound to form a Shiff base which is then reduced in the presence of asuitable reducing agent such as a borohydride derivative, e.g. sodiumborohydride or potassium borohydride, to give the desired R⁶ alkylresidue. As it is evident, the skilled man is capable of selecting thecarbonylic compound which, upon reduction, will give the desired alkylgroup represented by the symbol R⁶. The formation of the Shiff baseoccurs preferably in a polar aprotic solvent such as a lower alcohol,e.g. methanol or ethanol. The reaction is preferably conducted at about0° C. Also the reduction step is preferably conducted at about 0° C.,while is generally preferred to increase the temperature to at leastroom temperature to complete the reaction.

The following tables reports the physico-chemical parameters ofrepresentative examples of the compounds of the invention.

    TABLE I       COMPOUNDdi-amine reactant Formula.sup.(1)      ##STR27##      No. (RH) R R.sup.5 R.sup.6 (MW) C H N S       I NH.sub.2 CH.sub.2 CH.sub.2 NH.sub.2 NHCH.sub.2 CH.sub.2 NH.sub.2 H H C     .sub.89 H.sub.148 N.sub.22 O.sub.24 S.sub.4 52.45 7.32 15.12 6.29     (2038) 51.25 7.09 15.36 5.98 II NH.sub.2 (CH.sub.2).sub.4 NH.sub.2     NH(CH.sub.2 ).sub.4NH.sub.2.sup.(3) H H C.sub.91 H.sub.152 N.sub.22     O.sub.24 S.sub.4 52.90 7.41 14.91 6.21      (2066) 51.96 7.07 15.57 5.90     IIIa NH.sub.2 (CH.sub.2).sub.3 N(CH.sub.3).sub.2 NHCH.sub.2 CH.sub.2     CH.sub.2 N(CH.sub.3).sub.2 H H C.sub.92 H.sub.156 N.sub.22 O.sub.24     S.sub.4 53.06 7.55 14.80 6.16      (2083) 52.83 7.56 14.73 6.04 IIIb     NH.sub.2 (CH.sub.2).sub.3 N(CH.sub.3).sub.2 NHCH.sub.2 CH.sub.2 CH.sub.2     N(CH.sub.3).sub.2.HCl.sup.(4) H H C.sub.92 H.sub.157 ClN.sub.22 O.sub.24     S.sub.4 52.14 7.47 14.54 6.05      (2119) 52.20 7.49 14.61 5.91  IV      ##STR28##      ##STR29##      H H C.sub.91 H.sub.152 N.sub.23 O.sub.24 S.sub.4(2080) 52.5350.07     7.367.00 16.4815.73 6.166.57  V HN[CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2     ].sub.2      ##STR30##      H H C.sub.95 H.sub.161 N.sub.23 O.sub.24 S.sub.4(2137) 53.3852.84     7.597.25 15.0715.21 6.006.38      VI     ##STR31##      ##STR32##      H H C.sub.91 H.sub.150 N.sub.22 O.sub.24 S.sub.4(2064) 52.9552.58     7.327.14 14.9315.46 6.216.73      VII     ##STR33##      ##STR34##      H H C.sub.92 H.sub.152 N.sub.22 O.sub.24 S.sub.4(2079) 53.1553.13     7.367.15 14.8215.53 6.176.43      VIII     ##STR35##      ##STR36##      H H C.sub.96 H.sub.158 N.sub.22 O.sub.24 S.sub.4(2132) 54.0853.66     7.477.27 14.4515.00 6.026.61      IX     ##STR37##      ##STR38##      H H C.sub.98 H.sub.156 N.sub.22 O.sub.24 S.sub.4(2154) 54.6455.19     7.307.19 14.3014.68 5.956.23  ACTAGARDINE H H C.sub.87 H.sub.142     N.sub.20 O.sub.25 S.sub.4 -- -- -- --    (1996) 52.03 7.14 14.32 6.40 X  N     HCH.sub.2 CH.sub.2 NH.sub.2 CH.sub.2 CH.sub.2 OCH.sub.3 H C.sub.92     H.sub.154 N.sub.22 O.sub.25 S.sub.4 *      (2096) XI  NHCH.sub.2     CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2 CH.sub.3 H C.sub.93 H.sub.158     N.sub.22 O.sub.24 S.sub.4 *      (2097) XII  NHCH.sub.2 CH.sub.2     CH.sub.2 N(CH.sub.3).sub.2 C.sub.2 H.sub.5 H C.sub.94 H.sub.160 N.sub.22     O.sub.24 S.sub.4 *      (2111) XIII  NHCH.sub.2 CH.sub.2 CH.sub.2     N(CH.sub.3).sub.2 H C.sub.2 H.sub.5 C.sub.94 H.sub.160 N.sub. 22     O.sub.24 S.sub.4      (MW 2111)      XIV     ##STR39##      CH.sub.3 H C.sub.93 H.sub.154 N.sub.22 O.sub.24 S.sub.4 *(2093)  XV      ##STR40##      C.sub.2 H.sub.5 H C.sub.94 H.sub.156 N.sub.22 O.sub.24 S.sub.4 *(2107)     XVI      ##STR41##      CH.sub.3 H C.sub.97 H.sub.160 N.sub.22 O.sub.24 S.sub.4 *(2146)  XVII      ##STR42##      C.sub.2 H.sub.5 H C.sub.98 H.sub.162 N.sub.22 O.sub.24 S.sub.4     Notes to TABLE I:     *Elemental analysis confirms the assigned formula.     .sup.(1) The theoretical formulas of the derivatives were calculated     assuming the formula C.sub.87 H.sub.142 N.sub.20 O.sub.25 S.sub.4  for     actagardine. The compounds melted with decomposition over a range of     temperatures from 200° to 300° C.     .sup.(2) Analyses were carried out on the products dried at 150°     under nitrogen atmosphere. The purity of the derivatives (expressed as     percentages of the areas of the chromatographic peaks) was determined by     HPLC in reverse phase partition with a Waters chromatograph equipped with     pump mod. M45, Rheodyne valve mod. 7125 (20 μl loop), UV detector mod.     440 at 254 nm (1.10.sup.-2 UA) connected to data system SP 4000 (Spectra     Physics). The column (25 cm × 4.6 mm i.d.), prepacked with     Lichrosorb RP8 10 μm, was used at room temperature with 0.1 M phosphat     buffer pH 7.5/CH.sub.3 CN 60:40 (v/v) as the mobile phase and with a flow     rate of 1 ml/min.     Purity of the compounds according to this method was almost always higher     than 94%. In particular, the purity of derivative IIIb resulted 97%. The     inorganic residue found (after heating at 900° C.) in oxygen     atmosphere was always less than 0.5%.     .sup.(3) Purity (HPLC) ˜ 90%.     .sup.(4) Cl %: calculated 1.67, found 1.58

                                      TABLE II                                    __________________________________________________________________________              Solubility.sup.(1)         Partition Coeff..sup.(3)                           in water  Acid-base Titration.sup.(2)                                                                    (log P)                                  Compound  (mg/ml)   E W       pk.sub.2.sup.(4)                                                                     CH.sub.3 (CH.sub.2).sub.7 OH/H.sub.2                                          O                                        No.       pH 7.3                                                                             pH 4.0                                                                             HTBA HC10.sub.4                                                                         (MCS/H.sub.2 O)                                                                      H.sub.2 O.sup.(5)                                                                    pH 3.sup.(6)                      __________________________________________________________________________    I         20   80   2400 1154 9.7    -0.174 -0.973                            II        30   100  n.d. 1058 9.8    -0.468 -0.922                            IIIa      200  600  n.d. 1142 9.2    -0.790 -1.644                            IIIb      200  600  .sup.  1082                                                                        .sup.   2119.sup.(7)                                                               9.2    n.d.   -1.644                            IV        n.d. 100  n.d.  750 n.d.   -0.680 -1.353                            V         n.d. 200  n.d.  814 n.d.   -0.066 n.d.                              VI        <10  60   2289 1098 8.4    -0.133 -0.733                            VII       <10  45   2084 1036 7.5     0.214 -0.955                            VIII      <10  20   2160 1038 7.5     1.084 -0.562                            IX        <10  20   2037  905 7.2    n.d.   -0.145                            ACTAGARDINE                                                                             70   <10   985 1950 --     (-0.545).sup. (8)                                                                     0.357                            __________________________________________________________________________                         Acid-base Titration                                                           E W                                                      Compound No.                                                                           HTBA        HClO.sub.4  0.1 N NaOH                                                                            0.1 N HCl                            __________________________________________________________________________    X        (no titrable functions)                                                                   1068 (2 basic functions)                                 XI       "           1161 (2 basic functions)                                                                  --      --                                   XII      "           1198 (2 basic functions)                                                                  --      --                                   XIII     2074        1039 (2 basic functions)                                                                  2091    1052                                 XIV      (no titrable functions)                                                                   1065 (2 basic functions)                                                                  --      --                                   XV       "           1084 (2 basic functions)                                                                  --      --                                   XVI      "           1107 (2 basic functions)                                                                  --      --                                   XVII     "           1099 (2 basic functions)                                                                  --      --                                   __________________________________________________________________________     Notes to Table II:                                                            .sup.(1) Approximate values.                                                  .sup.(2) Acid-base titrations were carried out in both aqueous                [methylcellosolve (MCS): water 4:1 (v/v)] and nonaqueous [pyridine or         acetic acid] solvents. The pK.sub.MCS values (pK.sub.2) of the additional     basic functions were determined in MCS:H.sub.2 O 4:1 (v/v) solution by        titration of the compounds with 0.01 N Na OH. The presence of the free        amino group of actagardine (pK.sub.MCS 6.3) in the derivatives, except        compound XIII, was confirmed by titration with 0.01 N HCl. Compound XIII      did not show this pKa value but a pKa value between 8.9 and 9.4. The          equivalent weights (E W) were obtained by titration with either               hydroxytetrabutylamine (HTBA) in pyridine or perchloric acid (HClO.sub.4)     in acetic acid.                                                               .sup.(3) Partition coefficients (log P) were determined between noctanol      and water. The concentration of the compounds in each phase was determine     spectrophotometrically (UV).                                                  .sup.(4) All compounds possess also the ionizable basic function of           actagardine (pK.sub.MCS 6.3) unmodified. Titration in MCS:H.sub.2 O 4:1       (v/v) with 0.01 N HCl.                                                        .sup.(5) Distilled water.                                                     .sup.(6) 0.1 M acetate buffer pH 3.0.                                         .sup.(7) Values determined in MCS:H.sub.2 O 4:1 (v/v) by titration with       either 0.1 N NaOH or 0.1 N HCl.                                               .sup.(8) Actagardine (acid form) is not soluble in water. The value given     is log P between noctanol and 0.1 M phosphate buffer pH 7.3.                  n.d. = not determined.                                                        MCS = methylcellosolve;                                                  

                                      TABLE III                                   __________________________________________________________________________    Spectral data                                                                 __________________________________________________________________________           IR (v,cm.sup.-1).sup.(1)                                               Compound                    γC--N                                                                         νC--N    UV.sup.(2)                      No.    νN--H                                                                            amide I                                                                            amide II                                                                           amide III                                                                          (aromatic)                                                                          (piperazine)                                                                         νC--S                                                                          λmax,nm (E.sup.1%                                                      .sub.1 cm )                      __________________________________________________________________________    I      3280  1650 1525 1240 --    --     740 280 (21.00)                      II     3280  1650 1525 1240 --    --     740 280 (19.11)                      III (a,b)                                                                            3280  1650 1515 1235 --    --     735 a.282 (26.50)                                                                 b.279 (22.36)                    IV     3290  1655 1525 1240 --    --     740 279 (25.67)                      V      3290  1650 1525 1240 --    --     740 279 (25.04)                      VI     3300  1655 1530 1240 --    1030   740 280 (22.12)                      VII    3300  1655 1525 1235 --    1030,1000                                                                            740 280 (24.08)                      VIII   3300  1650 1530 1245 --    1030   740 280 (22.00)                      IX     3300  1655 1520 1235 700   1030,1000                                                                            740 278 (23.14)                      Actagardine                                                                          3300,3060                                                                           1660 1525 1235 --    --     740 278 (26.00)                      __________________________________________________________________________    (ν, cm.sup.-1).sup. (1)                                                    Compound                      νCO                                                                             γC--H                                                                        νC--N                              No.    νN--H                                                                           amide I                                                                             amide II                                                                            amide III                                                                           (ester)                                                                            (arom.)                                                                            (piperazine)                                                                          νC--S                      __________________________________________________________________________    X      3280 1650  1525  1240  1730 --   --      740                           XI     3280 1650  1515  1235  1730 --   --      735                           XII    3280 1650  1515  1235  1730 --   --      735                           XIV    3300 1655  1525  1235  1730 --   1030,1000                                                                             740                           XV     3300 1655  1525  1235  --   --   1030,1000                                                                             740                           XVI    3300 1650  1530  1245  1730 --   1030    740                           XVII   3300 1650  1530  1245  1730 --   1030    740                           __________________________________________________________________________     Notes to Table III:                                                           .sup.(1) IR spectra were recorded with a Perkin Elmer mod. 580                spectrophotometer in nujol mull.                                              .sup.(2) UV spectra were recorded with a PerkinElmer mod. 320 UVVIS           spectrophotometer in methanol solution.                                  

The compounds of the invention show antibacterial activity in vitro andin vivo. They are most active against S. mitis, S. salivarius, and S.pyogenes, including clinical isolates of these strains. They have, ingeneral, ameliorated activity compared to actagardine.

Minimal inhibitory concentration (MIC) are determined by the twofoldserial dilution method in Brain heart infusion broth (Difco); it issupplemented with 2% bovine serum when streptococci are tested. Theinoculum size is about 10³ colony units per ml (CFU/ml). The MIC isdefined as the lowest concentration which prevents visible growth afterovernight incubation at 37° C. The influence of serum is determined onS. aureus Tour by adding 30% bovine serum to the medium. Inoculum size(˜10⁶ CFU/ml) and 30% bovine serum did not influence the antibacterialactivity at a great extent; the MIC found in the presence of serum weregenerally lower than those without it. The antimicrobial activities ofthe compounds of the invention in comparison with reference compound(actagardine) are reported in the following Table IV:

                                      TABLE IV                                    __________________________________________________________________________    In vitro Anitbacterial Activity (MIC, μg/ml)                               __________________________________________________________________________                   COMPOUND                                                                                                     acta-                           MICROORGANISM  I  II III(a,b)                                                                           IV  V   VI VII                                                                              VIII                                                                             IX gardine                         __________________________________________________________________________    S. aureus TOUR                                                                         10.sup.3(1)                                                                         6.2                                                                              6.2                                                                              12.5 50  50  25 12.5                                                                             25 6.2                                                                              25                                       10.sup.6(1)                                                                         25 25 25   50  50  50 25 50 12.5                                                                             50                                       + serum.sup.(2)                                                                     3.1                                                                              3.1                                                                              6.2  12.5                                                                              12.5                                                                              25 12.5                                                                             12.5                                                                             6.2                                                                              25                              S. pyogenes                                                                            C 203 0.2                                                                              0.2                                                                              0.4  0.8 0.8 0.4                                                                              0.8                                                                              0.4                                                                              0.8                                                                              1.6                             S. pneumoniae                                                                          UC 41 12.5                                                                             12.5                                                                             6.2  12.5                                                                              25  6.2                                                                              6.2                                                                              6.2                                                                              6.2                                                                              25                              S. mitis L 1320.sup.(3)                                                                      3.1                                                                              3.1                                                                              3.1  3.1 6.2 3.1                                                                              0.8                                                                              1.6                                                                              0.8                                                                              12.5                            S. faecalis D                                                                          L 1321.sup.(3)                                                                      25 25 50   100 100 25 50 25 12.5                                                                             100                             S. viridans                                                                            L 1322.sup.(3)                                                                      50 50 50   25  100 50 12.5                                                                             50 6.2                                                                              100                             S. salivarius                                                                          L 1323.sup.(3)                                                                      0.4                                                                              0.4                                                                              0.8  0.8 6.2 0.4                                                                              0.05                                                                             0.8                                                                              0.05                                                                             3.1                             S. sanguis H                                                                           L 1324.sup.(3)                                                                      50 25 50   50  100 50 12.5                                                                             50 6.2                                                                              100                             S. bovis D                                                                             L 1325.sup.(3)                                                                      50 50 25   25  25  25 12.5                                                                             25 12.5                                                                             100                             __________________________________________________________________________                   COMPOUND                                                       MICROORGANISM  X   XI   XII XIII                                                                              XIV  XV  XVI XVII                             __________________________________________________________________________    S. aureus TOUR                                                                         10.sup.3(1)                                                                         >100                                                                              50   50  50  >100 100 100 100                                       10.sup.6(1)                                                                         >100                                                                              100  100 --  >100 >100                                                                              >100                                                                              >100                                      + serum.sup.(2)                                                                     >100                                                                              50   50  --  100  100 50  50                               S. pyogenes                                                                            C 203 --  1.6  3.1 0.8 6.2  6.2 3.1 3.1                              S. pneumoniae                                                                          UC 41 >100                                                                              50   25  12.5                                                                              100  12.5                                                                              50  50                               S. mitis L 1320.sup.(3)     3.1                                               S. faecalis D                                                                          L 1321.sup.(3)     100                                               S. viridans                                                                            L 1322.sup.(3) 50                                                    S. salivarius                                                                          L 1323.sup.(3)     0.8                                               S. sanguis H                                                                           L 1324.sup.(3)     50                                                S. bovis D                                                                             L 1325.sup.(3)     25                                                __________________________________________________________________________     Notes to Table IV:                                                            .sup.(1) Inoculum (CFU/ml)                                                    .sup.(2) In the presence of 30% bovine serum                                  .sup.(3) Clinical isolates.                                              

The antimicrobial activity of the compounds of the invention is alsoconfirmed in in vivo test in mices experimentally infected with S.pyogenes or S. pneumoniae. The experiments are conducted essentially asdescribed by R. Pallanza et al., J. Antimicrob. Chemother. 11, 419(1983).

The experimental infection was induced in mice by intraperitoneallyadministering a suspension of the test pathogens. Inocula had beenadjusted so that the untreated animals died of septicemia within 48 h.Animals were treated subcutaneously with the test compound once a dayfor three days starting about 30 min after infection.

The ED₅₀ value was calculated on the 10^(th) day by the method ofSpearman and Karber (D. J. Finney "Statistical Method in BiologicalAssay", Griffin, page 524, 1952) on the basis of the percentage ofsurvival at each dose. In the above conditions the ED₅₀ value of somerepresentative compounds of the invention are reported below:

    ______________________________________                                        Compound No.                                                                              Infecting Strain ED.sub.50 (mg/Kg)                                ______________________________________                                        I           S. pyogenes C 203    0.14                                         II          S. pyogenes C 203    0.19                                         III (b)     S. pyogenes C 203    0.47                                                     S. pneumoniae                                                                             UC41     3.5                                          IV          S. pyogenes C 203    0.62                                         V           S. pyogenes C 203    0.29                                         VI          S. pyogenes C 203    0.25                                         VII         S. pyogenes C 203    0.24                                         VIII        S. pyogenes C 203    0.22                                         IX          S. pyogenes C 203    0.2                                          XIII        S. pyogenes C 203    0.23                                         ACTAGARDINE S. pyogenes C 203    0.81                                         ______________________________________                                    

The antibacterial activity of compound III (a and b) was compared withthat of actagardine against growing cells of S. pyogenes C 203. CompoundIII shows a good bactericidal activity comparable to that ofactagardine, but at lower concentrations. For both antibiotics, 99% ofkilling effect was obtained after 5 h of incubation at doses 10 timesthe MIC, while 99.9% was reached in 24 h. For compound III, this levelof killing action was obtained also at a dose equal to the MIC (0.4ug/ml). This comparison was carried out on Todd-Hewitt broth(s)containing compound III and actagardine at concentrations equal to ormultiple of the MIC which were inoculated with growing cells of S.pyogenes C 203 (˜10⁶ CFU/ml). The cultures were incubated at 37° C. withshaking and viable cells were counted at intervals.

In view of the above the compounds of the present invention caneffectively be employed as the active ingredient of antimicrobialpreparations used in human and veterinary medicine for the preventionand treatment of infectious diseases caused by pathogenic bacteria whichare susceptible to said active ingredients. In such treatments, thesecompounds may be employed as such or in the form of mixtures in anyproportion. The compounds of the present invention can be administeredtopically or parenterally but, the parenteral administration ispreferred. Depending on the route of administration, these compounds canbe formulated into various dosage forms. For topical use the compoundsof the present invention may also be prepared in suitable forms forabsorption through the mucous membranes of the nose and throat orbronchial tissues and may conveniently take the form of liquid sprays orinhalants, lozanges, or throat paints. For medication of the eyes orears, the preparation may be presented in liquid or semi-liquid form.Topical applications may be formulated in hydrophobic or hydrophilicbases as ointments, creams, lotions, paints, or powders.

Compositions for injection may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Alternatively, the active ingredient may be in powder form forreconstitution at the time of delivery with a suitable vehicle, such assterile water.

The amount of active principle to be administered depends on variousfactors such as the size and condition of the subject to be treated, theroute and frequency of administration, and the causative agent involved.

The compounds of the invention are generally effective at a daily dosagecomprised between about 0.5 and about 30 mg of active ingredient per Kgof body weight, preferably divided in 2 to 4 administrations per day.

Particularly desirable compositions are those prepared in the form ofdosage units containing from about 20 to about 300 mg per unit.

Representative examples of preparation of pharmaceutical compositionsare as follows:

A parenteral solution is prepared with 100 mg of compound III dissolvedin 2 ml of sterile water for injection

A parenteral solution is prepared with 250 mg of compound III dissolvedin 3 ml of sterile water for injection

A topical ointment is prepared with

200 mg of compound III

3.6 g of polyethylene glycol 4000 U.S.P.

6.2 g of polyethylene glycol 400 U.S.P.

Besides their activity as medicaments, the compounds of the presentinvention can be used as animal growth promoters.

For this purpose, one or more of the compounds of the invention isadministered orally in a suitable feed. The exact concentration employedis that which is required to provide for the active agent in a growthpromotant effective amount when normal amounts of feed are consumed.

The addition of the active compounds of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compounds in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed.

The way in which such feed premixes and complete rations can be preparedand administered are described in reference books (such as "AppliedAnimal Nutrition", W. H. Freedman and Co., S. Francisco, USA, 1969 or"Livestock Feeds and Feeding", O and B Books, Corvallis, Oreg., USA,1977) and are incorporated herein by reference.

The following examples illustrate the manner in which the invention canbe practiced, but, as such, should not be construed as limiting itsoverall scope.

EXAMPLE 1

Preparation of compounds I-IIIa and V-IX (general procedure):

To a stirred solution of 1 mmol of actagardine and 4 mmol of the properdi-amine in 100 ml of DMF a solution of 2.5 mmol of DPPA in 20 ml of DMFis added dropwise in 30 min while cooling at 0°-5° C. The reactionmixture is kept 6 h at 5° C. and overnight at room temperature. Onadding 500 ml of ether a solid separates which is collected, washed with100 ml of ether, and re-dissolved in 500 ml of a mixturen-butanol:water:methanol 45:45:10 (v/v/v). The organic layer isseparated, washed with 100 ml of water and concentrated to a smallvolume under vacuum at 50° C. A solid is then precipitated by addingether; it is then collected and washed with ether.

1 g of this crude material is dissolved in 60 ml of a mixtureacetonitrile and 0.01M phosphate buffer pH 8, 85:15 (v/v), and theresulting solution is applied to a silica-gel (0.2-0.06 mm) column (200g), eluting with the following mixtures of acetonitrile and 0.01Mphosphate buffer pH 8 (v/v):

(a) 85/15 (0.2 l); (b) 80/20 (0.4 l); (c) 75/25 (0.8 l); (d) 70/30 (0.8l); (e) 65/35 (0.8 l)

Fractions of about 50 ml are collected and monitored by TLC using S.pyogenes C 203 to individuate the active fractions. Fractions containingthe active compound are pooled, one volume of n-butanol is added theretoand the solvents are evaporated under vacuum at room temperature until abutanolic solution residuates.

The residual butanolic solution is washed with water (three times) andthen concentrated to a small volume under vacuum. Upon standingovernight at room temperature, and cooling if necessary, a solidseparates which is collected washed with ether and dried under vacuumovernight at about 50° C.

The physico-chemical data of the obtained compounds are reported inTables I, II, and III.

EXAMPLE 2

Preparation of compound IV (actagardineN-(2-aminoethyl)-1,2-ethanediamine monocarboxamide):

(a) preparation of 1,7-dibenzylidenediethylenetriamine.

Four mmol of diethylenetriamine are reacted with benzaldehyde (12 mmol)in ethanol (100 ml) at room temperature.

When the reaction is completed generally the reaction mixture is cooled,concentrated to a small volume and the product which precipitates isrecovered by filtration.

(b) preparation of compound IV:

To a stirred solution of 1 mmol of actagardine and 4 mmol of1,7-dibenzylidenediethylenetriamine in 100 ml of DMF, a solution of 2.5mmol of DPPA in 20 ml of DMF is added dropwise in 30 min while coolingat 0°-5° C. The reaction mixture is kept 6 h at 5° C. and overnight atroom temperature. On adding 500 ml of ether a solid separates which iscollected, washed with 100 ml of ether, and re-dissolved in 500 ml of amixture n-butanol:water:methanol 45:45:10 (v/v/v). The organic layer isseparated, washed with 100 ml of water and concentrated to a smallvolume under vacuum at 50° C. A solid is precipitated by adding etherand is then collected, and washed with ether. 1 g of this crude materialis dissolved in 60 ml of a mixture acetonitrile and 0.01M phosphatebuffer pH 8, 85:15 (v/v), and the resulting solution is applied to asilica-gel (0.2-0.06 mm) column (200 g), eluting with the followingmixtures of acetonitrile and 0.01M phosphate buffer pH 8 (v/v):

(a) 85/15 (0.2 l); (b) 80/20 (0.4 l); (c) 75/25 (0.8 l); (d) 70/30 (0.8l); (e) 65/35 (0.8 l).

Fractions of about 50 ml are collected and monitored by TLC using S.pyogenes C 203 to individuate the active fractions.

The fractions which contain the active compound are pooled, one volumeof n-butanol is added thereto and the solvents are evaporated undervacuum at room temperature until a butanolic solution residuates.

The residual butanolic solution is washed with water (three times) andthen concentrated to a small volume. Upon standing overnight at roomtemperature, and cooling if necessary, a solid separates which iscollected washed with ether and dried under vacuum overnight at about50° C.

1 g of this product (which is the dibenzylidene derivative of thecompound of the title) is dissolved in 200 ml of a mixture of 0.1N HCl:DMF 9:1 (v/v) at room temperature with stirring. After standingovernight at room temperature, 250 ml of n-butanol is added, the pH ofthe aqueous layer is brought to 7.0 with 3% aqueous NaHCO₃ and theorganic layer is separated and concentrated to a small volume. On addingether, a solid separates which is collected, washed with ether, andpurified on a silica-gel (0.2-0.06 mm) column eluting withacetonitrile/0.01M phosphate buffer pH 8 as described above for thedibenzylidene derivative. The physico-chemical data are reported inTables I, II and III.

EXAMPLE 3

Alternative preparation of compound VIII:

To a stirred solution of 1 mmol of actagardine and 4 mmol ofcyclopentylpiperazine dihydrochloride in 100 ml of DMF 10 mmol oftriethylamine is added while cooling at about 0° C. Then a solution of2.5 mmol of DPPA in 20 ml of DMF is added dropwise in 30 min whilekeeping the temperature at 0°-5° C. The reaction mixture is then kept 6h at about 5° C. and overnight at room temperature. On adding 500 ml ofether a solid separates which is collected, washed with 100 ml of ether,and re-dissolved in 500 ml of a mixture n-butanol:water:methanol45:45:10 (v/v/v). The organic layer is separated, washed with 100 ml ofwater and concentrated to a small volume under vacuum at 50° C. A solidis then precipitated by adding ether and it is collected and washed withether. 1 g of this crude material is dissolved in 60 ml of a mixtureacetonitrile and 0.01M phosphate buffer pH 8, 85:15 (v/v), and theresulting solution is applied to a silica-gel (0.2-0.06 mm) column (200g), eluting with the following mixtures of acetonitrile and 0.01Mphosphate buffer pH 8 (v/v):

(a) 85/15 (0.2 l); (b) 80/20 (0.4 l); (c) 75/25 (0.8 l); (d) 70/30 (0.8l); (e) 65/35 (0.8 l).

Fractions of about 50 ml are collected and monitored by TLC using S.pyogenes C 203 to individuate the active fractions.

Fractions containing the active compound are pooled, one volume ofn-butanol is added thereto and the solvents are evaporated under vacuumat room temperature until a butanolic solution residuates.

The residual butanolic solution is washed with water (three times) andthen concentrated to a small volume.

Upon standing overnight at room temperature, and cooling if necessary, asolid separates which is filtered off washed with ether and dried undervacuum overnight at about 50° C.

The physico-chemical data of the obtained compound are reported inTables I, II and III.

EXAMPLE 4

Preparation of compound IIIb (actagardine3,3-dimethylamino-1-propylamide monocarboxamide, hydrochloride):

To a stirred solution of 1 mmol of IIIa in 200 ml of water, 10 ml of0.1N HCl is added dropwise while cooling at 5° C. The resultingsolution, brought to about pH 5, is extracted with 400 ml of n-butanol.

The organic layer is separated and concentrated to a small volume at 35°C. under vacuum. On adding ether, the solid product of the titleseparates which is collected, washed with ether, and dried under vacuumovernight at 50° C.

The physico-chemical data are reported in Tables I, II and III.

EXAMPLE 5

Preparation of compounds X-XII and XIV-XVII (general procedure):

To a stirred suspension of the corresponding basic amide (namely: Co.Ifor Co.X, Co.III for Co.XI-XII, Co.VII for Co.XIV-XV and Co. VIII forCo.XVI-XVII) (1 mmole) in 400 ml of the proper alcohol R⁵ OH, 37%hydrochloric acid (about 4 ml) is added while stirring at roomtemperature. The final alcoholic solution is about 0.1M alcoholic HCl.The reaction course is monitored by TLC (on silica gel plates; silicagel 60-F₂₅₄ Merck; mobile phase: n-butanol/acetic acid/water, 4:1:1(v/v/v). The reaction is generally completed in 24-96 h.

The resulting solution is concentrated under vacuum at 40°-50° C. Theoily residue is washed with acetone and triturated to obtain a solidwhich is collected by filtration and washed with a mixture acetone/ethylether, 1:1 (v/v) and then with ether. This solid is then dried undervacuum over KOH pellets at room temperature for 24-48 h.

The obtained compounds are the hydrochlorides of the compounds of thetitle (yield=0.85-0.95 mmol; 85-95%).

The corresponding analytical pure free bases are prepared by columnchromatography from the above hydrochlorides according to the followingprocedure: 1.5 g of the hydrochloride obtained above is dissolved in 60ml of a mixture acetonitrile/water 85:15 (v/v), the resulting solutionis adjusted to pH 6.5 with phosphate buffer and applied to a silica-gel(0.06-0.2 mm) column (200 g), eluting with a linear gradient from 15% to40% of water in acetonitrile, in 20 h at rate of 200 ml/h.

Fractions of about 25 ml are collected and monitored by TLC. Fractionscontaining the free base compound are pooled, about one volume ofn-butanol is added thereto and the solvents are evaporated under vacuumat room temperature until a butabolic solution residuates. The residualbutanolic solution is washed with water (three times) and thenconcentrated to a small volume under vacuum.

Upon standing overnight at room temperature, and cooling if necessary, asolid separates which is collected, washed with ether and dried in theair for 2-3 days. The physico-chemical data of the obtained compoundsare reported in Tables I, II, and III.

EXAMPLE 6

Preparation of compound XIII (N-ethyl actagardine3,3-dimethylamino-1-propylamide monocarboxamide).

To a stirred solution of 1 mmol of IIIa in 300 ml of metanol, 0.45 ml ofacetaldehyde is added dropwise while cooling to 0° C. The reactionmixture is stirred at 0° C. for 2 h, afterwards 0.3 g of NaBH₄ is addedportionwise in 1 h. Stirring is continued for additional 2 h at roomtemperature, then NHCl is added at about pH 4. The resulting solution ispoured into 900 ml of water while cooling at 5° C. After extraction with900 ml of n-butanol, the organic layer is separated, washed with 500 mlof water and concentrated to a small volume at 40° C. under vacuum. Onadding ether, the solid product of the title separates which iscollected, washed with ether, and dried under vacuum overnight at 40° C.Yield 1.47 g. The physico-chemical data are reported in Table I, II, andII.

We claim:
 1. Basic monoamide derivatives of actagardine of formula##STR43## wherein the group ##STR44## represents the actagardinenucleus, R represents the group ##STR45## wherein R¹ and R²independently represents hydrogen, a group of formula ##STR46## in whichn represents an integer from 2 to 8 and R³ and R⁴ independentlyrepresent hydrogen or (C₁ -C₄)alkyl or R³ and R⁴ taken togetherrepresent a --(CH₂)₃ --, --(CH₂)₄ --, --(CH₂)₂ --O--(CH₂)₂ --, --(CH₂)₂--S-- --(CH₂)₂ --, or --(CH₂)₅ -- group, or R¹ and R² taken togetherwith the adjacent nitrogen atom represent a piperazine moiety which maybe substituted in position 4 with a substituent selected from (C₁-C₄)alkyl, (C₅ - C₇)cycloalkyl, benzyl, and substituted benzyl whereinthe phenyl moiety bears 1 or 2 substituents selected from chloro, bromo,nitro, (C₁ -C₄)alkyl and (C₁ -C₄)alkoxy, R⁵ represents hydrogen, (C₁-C₄)alkyl, (C₁ -C₄)alkoxy (C₂ -C₄)alkyl, and R⁶ represents hydrogen or(C₁ -C₄)alkyl, with the proviso that R¹ and R² can not simultaneouslyrepresent hydrogen, and the acid and base addition salts thereof.
 2. Acompound according to claim 1 wherein R represents a group selectedfrom: ##STR47##
 3. A compound according to claim 1 wherein R representsa group selected from: ##STR48##
 4. A compound according to claim 1wherein R represents the group

    NHCH.sub.2 CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2

or a corresponding pharmaceutically-acceptable acid addition salt.
 5. Acompound according to claims 1, 2, 3, or 4 wherein R⁵ and R⁶ representhydrogen.
 6. A compound according to claims 1, 2, 3, or 4 wherein R⁵represents hydrogen and R⁶ represents (C₁ -C₄)alkyl.
 7. A pharmaceuticalcomposition which comprises a compound of claims 1, 2, 3 or 4 inadmixture with a pharmaceutically acceptable carrier.